Cell movement in response to specific stimuli is observed to occur in prokaryotes and eukaryotes (Doetsch R N and Seymour W F, Life Sciences, 1970, 9:1029–1037; Bailey G B et al., J Protozool, 1985, 32:341–346). Cell movement seen in these organisms has been classified into three types; chemotaxis or the movement of cells along a gradient towards an increasing concentration of a chemical; negative chemotaxis which has been defined as the movement down a gradient of a chemical stimulus and chemokinesis or the increased random movement of cells induced by a chemical agent. The receptors and signal transduction pathways for the actions of specific chemotactically active compounds have been extensively defined in prokaryotic cells. Study of E. Coli chemotaxis has revealed that a chemical which attracts the bacteria at some concentrations and conditions may also act as a negative chemotactic chemical or chemorepellent at others (Tsang N et al., Science, 1973, 181:60–69; Repaske D and Adler J., J Bacteriol, 1981, 145:1196–1208; Tisa L S and Adler J., Proc Natl Aca Sci U.S.A., 1995, 92:10777–10781; Taylor B L and Johnson M S., FEBS Lett, 1998, 425:377–381).
Accumulation of immune cells at sites of injury or infection is a critical dimension of host defense that is achieved by highly conserved mediators of cell adhesion and cell motility. The large family of protein cytokines capable of inducing cell migration is termed collectively, chemokines, which can be produced by virtually every cell type in mammals (Wells, T. N., et al., Trends Pharmacol Sci, 1998, 19:376–380; Baggiolini, M., Nature, 1998, 392:565–568; Luster, A. D., N Engl J Med, 1998, 338:436–445). Chemokines mediate their function via seven-transmembrane, G protein-coupled receptors (7-TMR); the absence of either chemokines or their receptors results in marked phenotypic alterations in mice (Luster, A. D., supra; Ma, Q., et al., Proc Natl Acad Sci USA, 1998, 95:9448–53; Ma, Q., et al., Immunity, 1999, 10:463–471). These include altered inflammatory responses to pathogenic or allergenic challenges and mitigated atherosclerotic changes in models of vascular disease (Ross, R., N Engl J Med, 1999, 340:115–26). Extracellular fluids at sites of injury or infection have been reported to contain high concentrations of calcium (Menkin, V., Biochemical mechanisms in inflammation, 1981, Charles Thomas Publisher, Illinois, USA; Lin, C-Y and Huang, T-P., Nephron, 1991, 59:90–95; Kaslick, R. S., et al., J Periodonto, 1970, 41:93–7), and chronic inflammatory conditions and atherosclerosis are associated with deposition of calcium salts (Ross, R., supra; Tanimura, A., et al., J Exp Pathol, 1986, 2:261–73; McCarty, D. J., Dis Mon, 1994, 40:253–299). The concentration of calcium in such settings can be substantially higher than that of the serum (Menkin, V., supra; Lin, C-Y and Huang, T-P., supra; Kaslick, R. S., et al., supra). We hypothesized that such extracellular calcium gradients actively participate in modulating the immune response, acting via the CaR.
The calcium-sensing receptor (CaR) is a member of the 7-TMR superfamily and is responsive to Ca++ concentrations within the millimolar range found in extracellular fluids (Brown, E. M., et al., Nature, 1993, 366:575–80) (SEQ ID NOs 1 and 2). It was originally defined by its role in mediating systemic calcium homeostasis; however, it has been subsequently shown to have pleiotropic effects including altering cellular proliferation, differentiation and apoptosis (Brown, E. M., et al., Vitamins and Hormones, 1999, 55:1–71; Lin, K. I., et al., Biochem Biophys Res Commun, 1998, 249:325–31; Freichel, M., et al., Endocrinology, 1996, 137:3842–8; McNeil, S. E., et al., J Biol Chem, 1998, 273:1114–20). In hematopoietic cells, it is expressed on mature monocyte/macrophages and subsets of progenitor populations in the bone marrow (House, M. G., et al., J Bone Min Res, 1997, 12:1959–1970; Yamaguchi, T., et al., Biochem Biophys Res Commun, 1998, 246:501–6). Animals engineered to be deficient in this receptor appear normal at birth, but die with severely elevated blood calcium levels within the first few weeks of life (Ho, C., et al., Nat Genet 1995, 11:389–94; Dutour, A., Eur J Endocrinol, 1996, 134: 542–3). Activation of the receptor is maximal at 5 mM Ca++ (Brown, E. M., et al., Vitamins and Hormones, 1999, 55:1–71), and selective CaR activators have been developed that efficiently mimic Ca++-induced activation through an allosteric mechanism (e.g., NPS R-467 and its less active stereoisomer, S-467) (Nemeth, E. F., et al., Proc Natl Acad Sci USA, 1998, 95:4040–5). These agents are low molecular weight compounds, termed “calcimimetics”, that interact with the CaR's transmembrane domains and potentiate the actions of polycationic agonists, such as Ca++ itself, which bind to the receptor's amino-terminal extracellular domain. Calcimimetics are currently in clinical trials for treating primary hyperparathyroidism, a disorder in which the CaR is underactive, and represent useful pharmacological tools for assessing the CaR's mediatory role in CaR-expressing cells in which high Ca++ modulates cellular function. CaR signal transduction is mediated via a pertussis toxin (PTX)-inhibitable Gαi pathway as well as a PTX-insensitive mechanism, likely involving Gαq/11 (Chen, C. J., et al., Endocrinology, 1989, 124:233–9; Varrault, A., et al., Endocrinology, 1995, 136:4390–6; Dare, E., et al., J Mol Endocrinol, 1998, 21:7–17).